Benzothiophenes to inhibit leukotrienes

ABSTRACT

A method of inhibiting leukotrienes comprising administering to a mammal in need thereof an effective amount of a compound having the formula ##STR1## wherein R 1  and R 2  are independently hydrogen or C 1  -C 6  alkyl; 
     R 3  is hydrogen, or a group of the formula ##STR2##  wherein R 4  is phenyl, substituted phenyl, naphthyl or substituted naphthyl, with the proviso that when R 1  and R 2  are both C 1  -C 6  alkyl, R 3  is not hydrogen; and pharmaceutically acceptable salts thereof.

BACKGROUND OF THE INVENTION

This invention relates to certain benzothiophenes, compositionscontaining those compounds, and methods of their use.

The enzyme 5-lipoxygenase (5-LO) catalyzes the first step of abiochemical synthesis pathway by which arachidonic acid is convertedinto leukotrienes. Numerous and extremely potent biological activitieshave been associated with leukotrienes. Leukotrienes have beenimplicated as important mediators in a variety of disease states such asasthma, arthritis, psoriasis, ischemia, allergy, adult respiratorydistress syndrome (ARDS), and inflammatory bowel disease (IBD).

Considerable efforts have been directed toward the control ofleukotriene biosynthesis. Generally, research efforts directed towardthe control of leukotriene biosynthesis have been directed toward thediscovery of inhibitors of the 5-LO pathway and, in particular, 5-LOspecific inhibitors.

In U.K. Patent Application GB 2,196,629 certain ringsubstituted-N-hydroxy-N-substituted benzamide and cinnamamide compoundsare disclosed as antileukotriene agents. The ring substituent may be agroup having the Formula (Ra) (Rb) C═CH-- where (Ra) (Rb)C═ is anunsaturated aliphatic hydrocarbylene group containing 3 to 19 carbonatoms, a group having the formula R₃ --C═C-- where R₃ is a hydrogen atomor a saturated or unsaturated aliphatic hydrocarbyl group containing 1to 18 carbon atoms or a group having the formula R₄ --S-- where R₄ is analiphatic hydrocarbyl group containing 1 to 20 carbon atoms. TheN-substituent may be a C₁ -C₆ alkyl group, a C₃ -C₇ cycloalkyl group ora substituted or unsubstituted aryl group.

In European Patent Application 0196184 certain aryl compounds aredisclosed which include, among many others, certain cinnamohydroxamicacid analogs and certain N-hydroxyureas in Examples 81-91. Certain ureabased or urea containing compounds that are said to inhibit lipoxygenaseare disclosed in EPO 0292699; EPO 0279281; and EPO 0279263. Thesereferences contain a recognition of the importance of 3-2-(halo-phenylthio)phenyl!prop-2-enyl subsituted on a urea skeleton.

In WO 90/12008 certain unsubstituted and substituted phenyl, naphthyland thienyl N-hydroxy ureas are disclosed as inhibitors of 5- and12-lipoxgenase. The preparation and biological activity for a number ofsuch derivatives is disclosed. The present invention is directed to thediscovery that a select group of N-hydroxy-N- 3- 2-4-halophenylthio)phenyl!-prop-2-enyl!ureas are extremely potent 5-LOinhibitors.

Another important compound of the thienyl N-hydroxyurea class is(I)-N-(1-benzo B!thien-2-ylethyl)-N-hydroxyurea, i.e., Zileuton. Thesynthesis of Zileuton is described in U.S. Pat. No. 4,873,259 and itsexperimental pharmacology and clinical evaluation are reviewed in "Drugsof the Future 1993", 18(7), P. 616-618.

The compounds of the present invention, as defined herein, areinhibitors of 5-LO and have useful medical prophylactic and therapeuticproperties.

SUMMARY OF THE INVENTION

This invention provides methods for inhibiting leukotrienes comprisingadministering to a mammal in need thereof an effective amount of acompound of formula I ##STR3## wherein R₁ and R₂ are independentlyhydrogen or C₁ -C₆ alkyl;

R₃ is hydrogen, or a group of the formula ##STR4## wherein R₄ is phenyl,substituted phenyl, naphthyl, or substituted naphthyl, with the provisothat when R₁ and R₂ are both C₁ -C₆ alkyl, R₃ is not hydrogen; andpharmaceutically acceptable salts thereof.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns the discovery that a group ofbenzothiophenes, those of formula I and II, are useful for inhibiting5-lipoxygenase and leukotrienes. Further, novel benzothiophenes offormula II are provided, below: ##STR5## wherein R₆ and R₇ areindependently hydrogen or C₁ -C₆ alkyl;

R₅ is naphthyl, substituted naphthyl, or phenyl substituted one to threetimes with C₁ -C₆ alkoxy, C₁ -C₆ alkyl, phenyl, or hydroxy; with theproviso that if the phenyl is substituted once with hydroxy, it mustfurther be substituted once or twice with C₁ -C₆ alkoxy, C₁ -C₆ alkyl,phenyl or hydroxy, and pharmaceutically acceptable salts thereof.

The therapeutic and prophylactic treatments provided by this inventionare practiced by administering to a human in need thereof a dose of acompound of formula I or II, or a pharmaceutically acceptable salt orsolvate thereof, that is effective to inhibit leukotrienes, or aphysiological disorder associated with leukotrienes.

The term "inhibit" includes its generally accepted meaning whichincludes prohibiting, preventing, restraining, and slowing, stopping orreversing progression, severity or a resultant symptom. As such, thepresent method includes both medical therapeutic and/or prophylacticadministration, as appropriate.

The term "substituted phenyl" and "substituted napthyl" includes theparticular aryl group substituted once to three times with C₁ -C₆ alkyl,C₁ -C₄ alkoxy, hydroxy, nitro, chloro, fluoro, or tri(chloro orfluoro)methyl.

A variety of physiologic functions have been associated withleukotrienes. As such, the compounds of formula I and II are believed tohave the ability to treat in mammals a variety of disorders associatedwith an excess of leukotrienes such as asthma and allergic diseases,(including allergic rhinitis and hayfever), eczema, bronchitis,inflammatory bowel disease, psoriasis, shock, ischemia, adultrespiratory distress syndrome and arthritis. Therefore, the presentinvention also provides methods of inhibiting the above disorders at therates set out, for inhibiting the 5-lipoxygenase conversion toleukotrienes, by administering to a mammal in need of 5-lipoxygenaseinhibition, an asthma, allergic disease, eczema, bronchitis,inflammatory bowel disease, psoriasis, shock, ischemia, adultrespiratory distress syndrome or arthritis relieving dose of a compoundof the present invention.

Recent clinical studies have supported a role for a leukotrieneantagonist in the treatment of asthma (Cloud et al. J. Allergy Clin.Immunol., 79 256 (1987)) thus providing further evidence of theimportance of leukotrienes in clinical asthma. Further evidence obtainedover the past few years has shown the presence of leukotrienes in sputumof patients with chronic bronchitis (Turnbull, et al., Lancet II, 526(1977) and cystic fibrosis (Cromwell, et al., Lancet II, 164 1981)),suggesting a role of leukotrienes in the pathology of those diseases aswell. Furthermore, Lewis and colleagues (Int. J. Immunopharmacology, 4,85 (1982)) have detected material in rheumatoid synovial fluid thatreacts antigenically with antibody to LTD₄. This may hallmark theexistence of leukotriene permeability factors that, together with LTB4,augment the inflammatory process in the diseased joints. Therefore, thecompounds described in this invention should also alleviate some of thesymptoms of chronic bronchitis and cystic fibrosis and possiblyrheumatoid arthritis by virtue of their ability to inhibit leukotrienes.The compounds are also useful for inhibiting the cardiovascular effectsof leukotrienes thereby rendering them useful for treating conditionssuch as shock and ischemic heart disease. Evidence that leukotrienes areinvolved in cardiovascular conditions and in shock syndromes is providedby the work of Cook et al., (J. Pharmacol. Exp. Ther., 235, 470-474(1985)); Eimerl et al., (Am. J. Physiol., 251 H700-H709 (1986)); Etemadiet al., (Circ. Shock, 22, 55-63 (1987)); and Hock and Lefer, (Circ.Shock, 17, 263-272 (1985)).

Generally, at least one compound of formula I or II is formulated withcommon excipients, diluents or carriers, and compressed into tablets, orformulated as elixirs or solutions for convenient oral administration,or administered by the intramuscular or intravenous routes. Thecompounds can be administered transdermally, and may be formulated assustained release dosage forms, aerosol forms, and the like.

The compounds used in the methods of the current invention can be madeaccording to established procedures, such as those detailed in U.S. Pat.Nos. 4,133,814, 4,418,068, and 4,380,635, all of which are incorporatedby reference herein. The following scheme and examples are provided toillustrate preparation of the compounds. ##STR6## The above Scheme I canbe carried out as a two-step process or a one-pot process. The followingdescribes the one pot process.

EXAMPLE 16-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(p-phenyl)phenylmethanone

To 100 mL of methylene chloride was added 2 drops DMF, 3 g (15 mmol) of4-phenyl benzoic acid, and 15 mls of SOCl₂. The mixture was heated toreflux for 16 hours then reduced to dryness. To the resulting acidchloride was added 4 g (15 mmol) of 6-methoxy-2-(4-methoxyphenyl)benzoB!thiophene and 600 mL of methylene chloride followed by 14 g (105 mmol)of AlCl₃ over 5 mins. This mixture was then heated to reflux for 45minutes, cooled to room temperature, and 15 mLs (203 mmol) of EtSH wasadded. The mixture was again heated to reflux for 35 minutes, cooled toroom temperature and quenched by careful addition of 50 mL MeOH followedby 100 mL of H₂ O. After the mixture turned yellow, the mixture waspartitioned. The organic layer was dried over Na₂ SO₄ and reduced todryness. The product was then purified over silica using a 15 to 30%gradient of EtOAc:hexanes as the eluent. The product was thencrystallized from hexanes to yield 300 mgs (11% yield) of the desiredproduct as a yellow solid. ¹ H NMR consistent with structure; FD+MS=422;EA (Theory/Found) C 76.76/75.94, H 4.29/4.83.

EXAMPLE 26-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(α-napthyl)methanone

As in Example 1, but using α-naphthoyl chloride as the startingmaterial. 700 mgs (48% yield) of product isolated as a yellow solid. ¹ HNMR consistent with structure; FD+MS=396; EA (Theory/Found) C75.74/76.00, H 4.07/4.25.

EXAMPLE 36-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(β-napthyl)methanone

As in Example 1, but using β-naphthoyl chloride as the startingmaterial. 810 mgs (55% yield) of product isolated as a bright orangesolid. ¹ H NMR consistent with structure; FD+MS=396; EA (Theory/Found) C75.74/75.76, H 4.07/4.36.

The following is a description of Scheme 1 as a two step synthesis.

EXAMPLE 46-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(p-phenyl)phenylmethanone

(a) To 800 mls of methylene chloride at 0 degrees C. was added 16.6 g(76.6 mmol) of p-phenyl benzoyl chloride, 13.8 g (51.1 mmol) of6-methoxy-2-(4-methoxyphenyl)benzo B!thiophene, and then 20.43 g (153mmol) of AlCl₃ was added portion wise over 20 minutes. The mixture wasstirred at 0 degrees for 2 hours then poured over ice and diluted withbrine. The layers were separated and the organic layer was washed ×3×350mls with saturated sodium bicarbonate solution followed by ×2×400 mlswith de-ionized water. The organic layer was dried on sodium sulfate andreduced to dryness. The crude product was purified using HPLC and a 5 to25% gradient of EtOAc:hexanes as the eluent to provide 14.5 g (63%yield) of product as a yellow solid. ¹ H NMR consistent with structure.

(b) To 100 mls of methylene chloride at 0 deg C. was added 6 g (13.3mmol) of the compound from step 4(a), 8.9 g (66.7 mmol) AlCl₃ was addedportion wise over 20 minutes, and then 10 mls (135 mmol) of EtSH wasadded all at once. This mixture was heated to reflux for 2.5 hours andthen allowed to cool back to room temperature. The reaction was thenquenched by carefully pouring over ice, adding 50 mls THF, then dilutingwith brine. The layers were separated and the organic layer was washed×3×350 mls with saturated sodium bicarbonate solution followed by ×1×500mls de-ionized water. The organic layer was then dried on sodium sulfateand reduced to dryness. The crude product was purified on silica using15 to 30% EtOAc:hexanes gradient as the eluent to yield 3.7 g of thedesired product as a yellow solid. ¹ H NMR consistent with structure;FD+MS=422.

EXAMPLE 5 6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(α-napthyl)methanone

(a) As in Example 4a, but using α-naphthoyl chloride as the startingmaterial. 10.6 g (68% yield) of product isolated as a yellow solid. ¹ HNMR consistent with structure; FD+MS=424.

(b) As in example (4b), but using the product from step 5(a) as thestarting material. 4.8 g (87% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure; FD+MS=396.

EXAMPLE 66-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3yl)(β-napthyl)methanone

(a) As in example 4(a), but using β-naphthoyl chloride as the startingmaterial. 9.2 g (59% yield) of product isolated as a yellow solid. ¹ HNMR consistent with structure; FD+MS=424.

(b) As in example 4(b), but using the product from step 6(a) as thestarting material. 5.3 g (97% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure; FD+MS=396.

EXAMPLE 7 (a)6-methoxyphenyl-2-(p-methoxyphenyl)-(benzo(b)thien-3-yl(4-hydroxy-3,5-dimethoxyhenyl)methanone(b)6-methoxyphenyl-2-(p-methoxyphenyl)-(benzo(b)thien-3-yl(3,4,5-trimethoxyphenyl)methanone

(c) As in example 4(a) but using 3,4,5-tri-methoxy benzoyl chloride asthe starting material. Both products were isolated from a silica columnusing 20 to 40% EtOAc:hexanes as the eluent.

Product (a) was isolated as a yellow solid, with a yield of 1.8 g (38%yield) ¹ H NMR consistent with structure; FD+MS=450; EA (Theory/Found) C66.65/66.86, H 4.92/5.21. Product (b) was isolated as a yellow solid,with a yield of 900 mgs (19% yield) ¹ H NMR consistent with structure;FD+MS=464; EA (Theory/Found) C 67.22/67.48, H 5.21/5.26.

EXAMPLE 86-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(b)thien-3-yl(p-t-butylphenyl)methanone

(a) As in example 4a, but using 4-t-butyl benzoyl chloride as thestarting material. 3.1 g (97% yield) of product isolated as a thickyellow oil. ¹ H NMR consistent with structure; FD+MS=430; EA(Theory/Found) C 75.32/75.49, H 6.09/6.23.

(b) As in example 4(b), but using the product from step 8(a) as thestarting material. 550 mgs (59% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure; FD+MS=402. EA (Theory/Found) C74.60/74.79, H 5.51/5.80.

EXAMPLE 96-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thienyl-3-yl(p-methylphenyl)methanone

(a) As in example 4, but using 4-methyl benzoyl chloride as the startingmaterial. 1.9 g (65% yield) of product isolated as a yellow solid. ¹ HNMR consistent with structure; FD+MS=388; EA (Theory/Found) C74.20/73.85, H 5.19/5.20.

(b) As in example 4(b), but using the product from Step 9(a) as thestarting material. 700 mgs (75% yield) of product isolated as a yellowsolid. hu 1H NMR consistent with structure; FD+MS=360. EA (Theory/Found)C 73.31/73.57, H 4.47, 4.58.

EXAMPLE 10 6-hydroxyphenyl-2-(p-hydroxy phenyl)-(benzo(B)thienyl-3-yl(m-methylphenyl)methanone

(a) As in example 4(a), but using m-methyl benzoyl chloride as thestarting material. 4.7 g (60% yield) of product isolated as a yellowgum. ¹ H NMR consistent with structure; FD+MS=388; EA (Theory/Found) C74.20/74.46, H 5.19/5.33.

(b) As in example 4(b), but using the product from step 10(a) as thestarting material. 190 mgs (20% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure. EA (Theory/Found) C73.31/73.08, H 4.47/4.70.

EXAMPLE 116-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl(m-hydroxyphenyl)methanone

(a) As in example 4(a), but using m-methoxy benzoyl chloride as thestarting material. 2.5 g (84% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure; FD+MS=404.

(b) As in example 4(b), but using the product of step 11(a) as thestarting material. 400 mgs (45% yield) of product isolated as a yellowsolid. ¹ H NMR consistent with structure; FD+MS=362. EA (Theory/Found) C69.60/68.61, H 3.89/4.44.

The compounds used in the methods of this invention may formpharmaceutically acceptable salts with a wide variety of organic andinorganic bases and include the physiologically acceptable salts whichare often used in pharmaceutical chemistry. Such salts are also part ofthis invention. Bases commonly used for formation of salts includeammonium hydroxide and alkali and alkaline earth metal hydroxides,carbonates, as well as aliphatic and primary, secondary and tertiaryamines, aliphatic diamines. Bases especially useful in the preparationof addition salts include ammonium hydroxide, potassium carbonate,methylamine, diethylamine, ethylene diamine and cyclohexylamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions.

Pharmaceutical formulations can be prepared by procedures known in theart. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of excipients, diluents,and carriers that are suitable for such formulations include thefollowing: fillers and extenders such as starch, sugars, mannitol, andsilicic derivatives; binding agents such as carboxymethyl cellulose andother cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; moisturizing agents such as glycerol; disintegrating agentssuch as calcium carbonate and sodium bicarbonate; agents for retardingdissolution such as paraffin; resorption accelerators such as quaternaryammonium compounds; surface active agents such as cetyl alcohol,glycerol monostearate; adsorptive carriers such as kaolin and bentonite;and lubricants such as talc, calcium and magnesium stearate, and solidpolyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration or as solutions appropriate forparenteral administration, for instance by intramuscular, subcutaneousor intravenous routes. Additionally, the compounds are well suited toformulation as sustained release dosage forms and the like. Also, thecompounds may be formulated for aerosol use or nasal inhalation. Theformulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

The particular dosage of a compound of formula I or II required toinhibit leukotrienes or a physiological disorder associated therewith,or to carry out any of the methods disclosed herein, according to thisinvention, will depend upon the severity of the condition, the route ofadministration, and related factors that will be decided by theattending physician. Generally, accepted and effective daily doses willbe from about 0.1 to about 1000 mg/day, and more typically from about 50to about 200 mg/day. Such dosages will be administered to a subject inneed thereof from once to about three times each day, or more often asneeded to effectively inhibit leukotrienes or their effects, or anyother use disclosed herein.

FORMULATIONS

In the formulations which follow, "Active ingredient" means a compoundof formula I or II.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active ingredient 0.1-1000    Starch, NF        0-650    Starch flowable powder                      0-650    Silicone fluid 350 centistokes                      0-15    ______________________________________

The ingredients are blended, passed through a No. 45 mesh U.S. sieve,and filled into hard gelatin capsules.

Examples of specific capsule formulations that may be made include thoseshown below:

Formulation 2: Capsule

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active Ingredient 1    Starch, NF        112    Starch flowable powder                      225.3    Silicone fluid 350 centistokes                      1.7    ______________________________________

Formulation 3: Capsule

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active Ingredient 5    Starch, NF        108    Starch flowable powder                      225.3    Silicone fluid 350 centistokes                      1.7    ______________________________________

Formulation 4: Capsule

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active Ingredient 10    Starch, NF        103    Starch flowable powder                      225.3    Silicone fluid 350 centistokes                      1.7    ______________________________________

Formulation 5: Capsule

    ______________________________________    Ingredient        Quantity (mg/capsule)    ______________________________________    Active Ingredient 50    Starch, NF        150    Starch flowable powder                      397    Silicone fluid 350 centistokes                      3.0    ______________________________________

The specific formulations above may be changed in compliance with thereasonable variations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 6: Tablets

    ______________________________________    Ingredient       Quantity (mg/tablet)    ______________________________________    Active ingredient                     0.1-1000    Cellulose, microcrystalline                     0-650    Silicon dioxide, fumed                     0-650    Stearate acid    0-15    ______________________________________

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.1-1000 mg of active ingredientare made up as follows:

Formulation 7: Tablets

    ______________________________________    Ingredient          Quantity (mg/tablet)    ______________________________________    Active ingredient   0.1-1000    Starch              45    Cellulose, microcrystalline                        35    Polyvinylpyrrolidone                        4    (as 10% solution in water)    Sodium carboxymethyl cellulose                        4.5    Magnesium stearate  0.5    Talc                1    ______________________________________

The active ingredient, starch, and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders which are thenpassed through a No. 14 mesh U.S. sieve. The granules so produced aredried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. Thesodium carboxymethyl starch, magnesium stearate, and talc, previouslypassed through a No. 60 U.S. sieve, are then added to the granuleswhich, after mixing, are compressed on a tablet machine to yieldtablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose aremade as follows:

    ______________________________________    Ingredient           Quantity (mg/5 ml)    ______________________________________    Active ingredient    0.1-1000   mg    Sodium carboxymethyl cellulose                         50         mg    Syrup                1.25       mg    Benzoic acid solution                         0.10       mL    Flavor               q.v.    Color                q.v.    Purified water to    5          mL    ______________________________________

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor, and color are diluted withsome of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

EXAMPLE 9

    ______________________________________    Ingredient        Quantity (mg/5 ml)    ______________________________________    Active ingredient  10 mg    Ethanol            50 mg    Dichlorodifluoromethane                      658 mg    (Propellant 12)    Dichlorotetrafluoroethane                      282 mg    (Propellant 114)    ______________________________________

The active ingredient is dissolved in the ethanol. The concentrate isfilled into extruded aluminium cans for inhalation aerosols. The cansare degassed with propellant 12 and sealed with an appropriate metereddose valve. The volume of product expelled per actuation is 50 to 100 μlequivalent to 0.5-1 mg active ingredient.

EXAMPLE 10

    ______________________________________    Aerosol             mg/ml    ______________________________________    Active ingredient   0.2    Sorbitan trioleate  0.27    Trichlorofluoromethane                        70.0    Dichloridifluoromethane                        280.0    Dichlorotetrafluoroethane                        1094.0    ______________________________________

Test Procedures

The following assays indicate the utility of the compounds.

5-Lipoxygenase (5-LPO) Assay

To determine the effect of compounds on 5-lipoxygenase activity,peripheral blood neutrophils are separated by density centrifugation asdescribed (Marder et al., Prostaglandins Leukot. and Essent. Fatty Acids46, 265-270, 1992) and the cytosolic 5-lipoxygenase is prepared fromisolated neutrophils according to a modification of Carter et al., (J.Pharmacolo Exp. Thero 256, 929-937 (1991)). Briefly, neutrophils arewashed twice in buffer (in millimolar: PIPES, 10; BES, 10; and EDTA, 1;pH 6.8) and diluted to 2.5×10⁷ cells/ml. Cells are disrupted withsonication and the cytosolic fraction obtained by centrifugation at20,000×g for 20 min. at 4° C. The resultant supernatant containing thecytosolic 5-lipoxygenase activity is stored frozen at -70° C. prior touse. Compounds are evaluated for 5-lipoxygenase inhibitory activity.Varying concentration of compounds in 200 ml aliquots containing 50 mlof human 20,000×g supernatant in assay buffer (in millimolar: PIPES, 10;BES, 10; EDTA, 1; NaCl, 100; ATP, 1.56; and CaCl₂, 2.5; pH 6.8)containing 3.0 mM AA are used. The reaction proceeds for 5 minutes at37° C. and is terminated upon the addition of 50 ml EDTA (250 mM). Thepercent inhibition of leukotriene B₄ is determined for eachconcentration of test compound as compared to vehicle (containing nocompound) control. The concentration representing 50% inhibition (IC₅₀)is determined by standard mathematical techniques. Leukotriene B₄ isdetermined by EIA. EIA reagents were purchased from Caymen Chemical Co.(Ann Arbor, Mich.) and used according to the assay procedures asoutlined therein.

cPLA2 Enzymatic Activity Assay

The substrate, sonicated liposomes containing 1-palmitoyl-2 ¹⁴C!arachidonoyl-sn-glycerol-3-phosphocholine ( ¹⁴ C!PC, 55 mCi/mmol fromNEN Research Products) and sn-1,2-dioleoylglycerol (DG, Avanti PolarLipids, Birmingham, Ala.) at a molar ratio of 2:1, are prepared asfollows. ¹⁴ C!PC (20 nmol, 1×10⁶ dpm, 50 uCi/ml in toluene/ethanol) andDG (10 nmol, 100 ug/ml in chloroform) are dried under nitrogen. Thelipids are dispersed in 1 ml of 150 mM NaC₁, 50 mM Hepes, pH 7.5 (assaybuffer) by sonication at 4° C., with Microson probe-sonicator (HeatSystems Ultrasonics) for 4×15 seconds, with 45 second intervals. Bovineserum albumin (essentially fatty acid free, from a 100 mg/ml stock inwater, Sigma) is added to a final concentration of 4 mg/ml. Samples tobe assayed for cPLA₂ activity are incubated with 50 ul liposomes (0.5nmol ¹⁴ C!PC, 50,000 dpm containing 0.25 nmol of DG) in a total volumeof 0.2 ml of assay buffer containing 1 mM CaCl₂ and 1 mM 2-ME.Incubations are carried out at 37° C. for 15 minutes and terminated byadding 2 ml of Dole's reagent (2-propanol/heptane/0.5M sulfuric acid,40:10:1 containing 10 ug/ml of stearic acid). After mixing, 1.2 ml ofheptane and 1 ml of water are added. The mixtures are briefly vortexedand the upper phase transferred to tubes containing 2 ml of heptane and150 mg of Bio-Sil (Bio-Rad Laboratories) activated at 130° C. beforeuse. The tubes are thoroughly vortexed and centrifuged (1000×g for 5minutes). The supernatants are decanted into scintillation vials. Afteraddition of 10 ml of a liquid scintillation cocktail (Ready Protein+,Beckman) radioactivity is counted using a Beckman liquid scintillationcounter Model LS 7000. High radioactive counts correlate with enzymaticactivity. The chromogenic cPLA2 enzyme assay used is analogous toReynolds et al., Analytical Biochemistry, 217, 25-32 (1194).

Inhibition of Eicosanoid Production by Plasma-depleted Blood.

Compounds are evaluated for their ability to inhibit production ofleukotriene B₄ (LTB₄) and thromboxane B₂ (TxB₂) upon stimulation ofhuman plasma-depleted blood with formyl-methionyl-leucyl-phenylalanine(FMLP) and thrombin. LTB₄ is produced by neutrophils and TxB₂ byplatelets stimulated at their respective receptors with FMLP andthrombin. The assay is carried out in 1 ml deep well polypropylene96-well plates (Beckman). For the tests ten ml of blood is collected inEDTA at a final concentration of 1.5 mg/ml. Then, 40 ml glucosephosphate buffer containing 0.1% gelatin is added and the suspensioncentrifuged at 900 g for 10 minutes at room temperature. The supernatantis discarded and the cells washed once with 50 ml buffer beforeresuspending them in 50 ml Krebs-Ringers-Henseleit Buffer containing0.1% gelatin, 1 mM CaCl₂, 1.1 mM MgCl₂ (KRH buffer). Working solutionsof test compounds are made by dissolving the material in DMSO at 10 mMand diluting with appropriate amounts of KRH buffer. An agonist solutionis made containing 1 μM FMLP (sigma) and 10 units/ml of thrombin in KRHbuffer (Enzyme Research Labs). The blood cell suspension is first warmedto 37° C. for 5 minutes and then mixed with cytochalasin B (sigma) at afinal concentration of 2 μg/ml. In each well, 200 μl of the compoundsolution is incubated with 250 μl of the blood cell, cytochalasin Bsuspension at 37° C. for 10 min. Stimulation of eicosanoid production isbegun by adding 50 μl of the agonist solution. The mixture is thenincubated for 2 min. before stopping the reaction by adding 50 μl of asolution containing 275 mM EDTA, 110 μM indomethacin (sigma) and 110 μMof the 5-lipoxygenase inhibitor, N-hydroxy-N- 3-2-(2-propynylthio)phenyl!-2-propenyl!urea. Subsequently, the plate iscentrifuged and an aliquot of the supernatant fluid removed for analysisof the eicosanoid content. The amount of LTB₄ and TxB₂ produced isdetermined by the use of a competitive enzyme immunoassay usingcommercial reagents from Cayman Chemical.

MCII MAST CELL LTC4 ASSAY

This assay measures the production of LTC4 by the cytokine dependentmast cell line MCII, stimulated by cross linking the high affinity Fcreceptor for IgE (Fce.sub.ε R1).

The bone marrow derived, cytokine dependent mouse mast cell line MCII(Lilly Research Laboratories) is maintained in log phase growth incomplete Dulbecco's' Modified Eagles Medium (GIBCO BRL) supplementedwith fetal bovine serum (Hyclone), gentamicin, penicillin andstreptomycin (GIBCO BRL) and using concanavalin A (ICNImmunoBiologicals) induced culture supernatants from the mouse helper Tcell clone D10.G4 (ATCC TIB 224) as a source of mast cell growthfactors. The MCII cells are passively sensitized with mouse monoclonalIgE by incubation with an appropriate dilution of IgEL b4 (ATCC TIB 141)antibody ascites at 37° C. for 60 minutes and then washed with Hank'sBalanced Salt Solution (GIBCO BRL) to remove unbound IgE. The sensitizedcells are then preincubated with drug in complete media for 10 minutesat 37° C. in 96 well polypropylene plates (Costar). Each drugconcentration is tested in triplicate at half log dilutions. The drugvehicle is dimethyl sulfoxide which is present at a final concentrationof 0.32% in all wells.

Following preincubation with drug the cells are stimulated by additionof an appropriate dilution of rat anti-mouse IgE monoclonal antibodyascites EM95 (Robert Coffman, DNAX Research Institute) to cross linkFce.sub.ε R1 to which IgE has bound. There are 40,000 MCII cells perwell in a final volume of 200 μl complete medium. The stimulated cellsare incubated at 37° C. for 10 minutes and then LTC4 production isstopped by addition of 25 μl of 180 mM EDTA to chelate extracellularCa⁺⁺.

The amount of LTC₄ produced is determined in a competitive enzymeimmunoassay (EIA) using commercial reagents purchased from CaymanChemical Co. In the EIA, free LTC4 in the sample competes with enzymeconjugated LTC4 for binding to immobilized anti-LTC4 antibody. Theenzyme conjugated to LTC4 is acetylcholinesterase and Elman's reagent isthe substrate for the enzyme. The concentration of LTC4 in the unknownsample is determined using a 4-parameter quadratic analysis of astandard curve generated by measuring the optical density resulting fromthe addition of known amounts of LTC4 to the EIA. The LTC4 captureantibody has a 46% cross reaction with LTD4 and 2% with LTE4.

The positive control on each plate is stimulated MCII cells withdimethyl sulfoxide vehicle alone and the negative control is the samereaction mixture without cells. The background negative control value issubtracted from experimental and positive control values and the dataare reported as percent of positive control. When the cells are notsensitized with IgE or in the absence of stimulation with rat anti-mouseIgE monoclonal antibody EM95, the production of LTC4 is below the limitof EIA detection at this sample volume.

MCII MAST CELL TOXICITY ASSAY

This toxicity assay is run in parallel with the MCII mast cell LTC4assay and serves as an indicator of compound effects on the viabilityand metabolic activity of the MCII cells. This "toxicity" assay is basedon the ability of dehydrogenase enzymes present in viable metabolicallyactive cells to bioreduce the tetrazolium salt XTT yielding a colored,water soluble formazan product. (see, Roehm et al.) "An improvedcolorimetric assay for cell proliferation and viability utilzing thetetrazolium salt XTT. J. Immunol. Methods 1422:257-65 (1991).

A duplicate polystyrene 96-well tissue culture plate (Costar) is set upwith sensitized MCII cells, EM95 ascites, media and dilutions of thetest compound as described above. The complete media is supplementedwith concanavalin A induced culture supernatants from the mouse helper Tcell clone D10.G4, as a source of mast cell growth factors.

Wells of the toxicity assay plate are pulsed with 25 μl XTT (Sigma) plusphenazine methosulfate (Aldrich) giving a final concentration of 0.2mg/ml XTT and 25 μM phenazine methosulfate. The plates are incubated at37° C. in 10% CO2 for 4-12 hours and then production of the XTT formazanproduct determined by measuring the optical density of the wells at awavelength of 450 nm. The positive control on each plate is stimulatedMCII cells with dimethyl sulfoxide vehicle alone and the negativecontrol is the same reaction mixture without cells. The backgroundnegative control value is subtracted from experimental and positivecontrol values and the data are reported as percent of positive control.

The results of the Assays are provided in the following Tables. (Theresults are in IC₅₀ μM). (Compound A is a compound of formula II whereR₆ and R₇ are hydrogen, and R₅ is phenyl. Compound B is a compound offormula I where R₁, R₂ and R₃ are hydrogen. Compound C is a compound offormula II wherein R₆ and R₇ are methyl, and R₅ is p-methoxyphenyl.Compound D is a compound of formula II wherein R₁ and R₂ are hydrogen,and R₅ is p-hydroxyphenyl.)

                  TABLE 1    ______________________________________    5-LPO Assay    Compound          IC.sub.50    ______________________________________    Example 2         1.7 ± 0.9 (n = 3)    Example 3         1.0 ± 0.5 (n = 3)    Compound B        0.2 ± 0.007 (n = 4)    Compound C        >10 (n = 4)    Zileuton          1.3 ± 0.9 (n = 3)    ______________________________________

                  TABLE 2    ______________________________________    C-PLA2 Enzyme    Compound      C14PC/DA6  Chromogenic    ______________________________________    Compound A    7.3 ± 0.5                             --                  (n = 3)    Compound B      7 ± 0.8                             >200                  (n = 3)    (n = 1)    Example 2     4.8 ± 1.6                             >200                  (n = 3)    (n = 1)    Example 3     2.3 ± 0.6                              122                  (n = 3)    (n = 1)    Example 1     <1.2        146                  (n = 2)    (n = 1)    ______________________________________

                  TABLE 3    ______________________________________    MCII Mast Cell    Compound        LTC.sub.4                             Toxicity    ______________________________________    Compound A      7.1 ± 0.5                             0.3                    (n = 2)  (n = 1)    Compound B      0.7      <10                    (n = 2)  (n = 1)    Example 2       0.5 ± 0.2                             >10                    (n = 4)  (n = 3)    Example 3       3.9 ± 1.8                             >10                    (n = 8)  (n = 16)    Example 1       4.4      >10                    (n = 2)  (n = 1)    Compound C      6.7 ± 3.0                    (n = 4)    Example 11      2.7    Example 10      1.1 ± 0.2                    (n = 3)    Example 8       3.5 ± 2.1                    (n = 3)    Example 9       0.8 ± 0.5                    (n = 3)    Example 7A      1.5 ± 0.3                    (n = 3)    Example 7B      1.5 ± 0.4                    (n = 3)    Zileuton        0.5 ± 0.3                    (n = 2)    ______________________________________

                  TABLE 4    ______________________________________    Plasma DPLTD Blood    Compound        LTB.sub.4                             TxB.sub.2    ______________________________________    Compound A      0.5      4.9                    (n = 2)  (n = 2)    Compound B      0.8      3.3                    (n = 2)  (n = 2)    Example 2       0.2 ± 0.1                             4.9 ± 2.4                    (n = 7)  (n = 6)    Example 3       0.2      1.4                    (n = 2)  (n = 2)    Example 1       2.2 ± .4                             2.4 ± 1.2                    (n = 3)  (n = 3)    Compound C      1.5 ± 0.4                             2.0 ± 0.6                    (n = 4)  (n = 4)    Compound D      1.6      >9.8                    (n = 2)  (n = 2)    ______________________________________

We claim:
 1. A method of inhibiting leukotrienes comprisingadministering to a mammal in need thereof an effective amount of acompound of formula I ##STR7## wherein R₁ and R₂ are independentlyhydrogen or C₁ -C₆ alkyl;R₃ is hydrogen, or a group of the formula##STR8## wherein R₄ is phenyl, substituted phenyl, naphthyl orsubstituted naphthyl with the proviso that when R₁ and R₂ are both C₁-C₆ alkyl, R₃ is not hydrogen; and pharmaceutically acceptable saltsthereof.
 2. The method of claim 1 wherein R₁ and R₂ are hydrogen and R₃is hydrogen.
 3. The method of claim 1 wherein R₃ is a group of theformula ##STR9##
 4. The method of claim 3 wherein R₄ is naphthyl,substituted naphthyl, (p-phenyl)phenyl, dimethoxyphenyl,trimethoxyphenyl, p-t-butylphenyl, p-methylphenyl, m-methylphenyl, orm-hydroxyphenyl.
 5. The method of claim 1 wherein said mammal suffersfrom asthma, arthritis, allergy, bronchitis, inflammatory bowel disease,psoriasis, eczema, shock, adult respiratory distress syndrome orischemia.
 6. The method of claim 1 wherein said compound is selectedfrom the group consistingof6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(p-phenyl)phenylmethanone;6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(α-napthyl)methanone;6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl)(β-napthyl)methanone;6-methoxyphenyl-2-(p-methoxyphenyl)-(benzo(B)thien-3-yl(4-hydroxy-3,5-dimethoxyphenyl)methanone;6-methoxyphenyl-2-(p-methoxyphenyl)-(benzo(B)thien-3-yl(3,4,5-trimethoxyphenyl)methanone;6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl(p-t-butylphenyl)methanone;6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thienyl-3-yl(p-methylphenyl)methanone;6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thienyl-3-yl(m-methylphenyl)methanone; or6-hydroxyphenyl-2-(p-hydroxyphenyl)-(benzo(B)thien-3-yl(m-hydroxyphenyl)methanone; and acceptable salts thereof.